The summary of last year's progress report was: In summary, we have identified asparagine as a key nutrient for UOK262 and UOK269 cells. Identification of an additional key nutrient was the goal for this year as set out in last year's report. To determine the effects of asparagine on gene expression, we have collected RNA for an RNASeq analysis of both UOK262 deplete and repleted cells after 96 hours exposure to glutamine, asparagine or both compared to cells grown without either amino acid supplementation. We chose 96 hours because that was the time point where the growth curves began to diverge. The RNA samples are now at FNLCR and will be available in about 1 month. This past year was devoted to following-up on the RNAseq experiment. Although we hypothesized that we would find stimulation of the MTOR signaling pathway, what we found instead was clear evidence that treatment the UOK262 FH- cells with G+A increases expression of a key elements of UPR (unfolded protein response) and hypoxia-inducible pathways compared to FH+ cells, after 96 hrs. RNAseq showed upregulation of DDIT3 (CHOP), GRP78, HSP90B (GRP94), DUSP1, JUNB, and ADM1 in the condition of G+A, but not in +G or +A, and only in UOK262 FH- cells and not in UOK262 FH+ cells. These were all confirmed by quantitative RT-PCR. These genes are associated with the induction of the Unfolded Protein Response (UPR). Using an orthogonal approach to confirm whether the UPR pathway was activated by +G+A but not by +G or +A, we looked at the ratio of XBP1 splicing, where UPR stimulation should increase the ratio of spliced to total XBP1 mRNA, and we found that the XBP1 splicing ratio increased by 3000-fold in UOK262 FH- in the G+A+ condition, but only 35-fold in the +G condition, not increased in the +A condition, and not increased in any condition in the UOK262 FH+ cell line. These findings confirm UPR induction in the UOK262 FH- cells that is dependent upon asparagine. To determine whether the cytotoxicity of other inducers of UPR would be affected by asparagine, we looked at the cytotoxicity of Brefeldin A (BFA), thapsigargin and tunicamycin in the presence and absence of asparagine. Asparagine sensitized UOK262 cells to BFA but not to thapsigargin or tunicamyicin. Later this year we plan 1) to perform radioisotope tracer (SIRM) experiments with labeled asparagine in the presence and absence of glutamine and labeled glutamine in the presence and absence of asparagine in UOK262 cells to determine how these two amino acids are metabolized and to possibly confirm our initial conclusion that these amino acids are channeled into separate metabolic pathways in these cells; and 2) using other agents that disrupt endoplasmic reticulum function in addition to BFA, we will start to identify the key metabolic pathways induced by asparagine in these cells and how the asparagine-induced UPR stimulates survival until it is overwhelmed and leads to cell death.